Cleavable linkers for polynucleotides

ABSTRACT

In various embodiments of the invention, novel compositions having a polynucleotide bound to a substrate via a cleavable linker are provided, and methods of cleaving a polynucleotide from a substrate are provided.

RELATED APPLICATIONS

Related subject matter is disclosed in U.S. patent application Ser. No.11/387,388 filed by Dellinger et al. entitled “Monomer Compositions forthe Synthesis of Polynucleotides, Methods of Synthesis, and Methods ofDeprotection”; U.S. patent application Ser. No. 11/388,112 filed byDellinger et al. entitled “Monomer Compositions for the Synthesis ofPolynucleotides, Methods of Synthesis, and Methods of Deprotection”;U.S. patent application Ser. No. 11/387,269 filed by Dellinger et al.entitled “Solutions, Methods, and Processes for Deprotection ofPolynucleotides”; U.S. Patent Application 60/785,130 filed by Dellingeret al. entitled “Use of Mildly Basic Solutions of Peroxyanions for thePost-Synthesis Deprotection of RNA Molecules and Novel MonomerCompositions for the Synthesis of RNA”; U.S. patent application Ser. No.11/751,692 filed by Dellinger et al. entitled “Thiocarbonate Linkers forPolynucleotides”; U.S. patent application Ser. No. 11/388,339 filed byDellinger et al. entitled “Phosphorus Protecting Groups”; allabove-mentioned patent applications filed on the same day as the presentapplication. Related subject matter is also disclosed in U.S. PatentApplication filed on Oct. 31, 2005 by Dellinger et al. entitled “Methodsfor Deprotecting Polynucleotides” having Ser. No. 60/731,723.

DESCRIPTION Field of the Invention

The invention relates generally to nucleic acid chemistry. Moreparticularly, the invention relates to providing a cleavable linker forrelease of polynucleotides, e.g. from a substrate. The invention isuseful in the manufacture of reagents and devices used in the fields ofbiochemistry, molecular biology and pharmacology, and in medicaldiagnostic and screening technologies, as well as other uses.

Background of the Invention

Solid phase chemical synthesis of DNA fragments is routinely performedusing protected nucleoside phosphoramidites. Beaucage et al. (1981)Tetrahedron Lett. 22:1859. In this approach, the 3′-hydroxyl group of aninitial 5′-protected nucleoside is first covalently attached to thepolymer support. Pless et al. (1975) Nucleic Acids Res. 2:773. Synthesisof the oligonucleotide then proceeds by deprotection of the 5′-hydroxylgroup of the attached nucleoside, followed by coupling of an incomingnucleoside-3′-phosphoramidite to the deprotected hydroxyl group.Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185. The resultingphosphite triester is finally oxidized to a phosphotriester to completeone round of the synthesis cycle. Letsinger et al. (1976) J. Am. Chem.Soc. 98:3655. The steps of deprotection, coupling and oxidation arerepeated until an oligonucleotide of the desired length and sequence isobtained. Optionally, after the coupling step, the product may betreated with a capping agent designed to esterify failure sequences andcleave phosphite reaction products on the heterocyclic bases.

Solid phase polynucleotide synthesis results in a polynucleotide boundupon a solid support. Typically, an additional step releases thepolynucleotide from the solid support after the polynucleotide strandhas been synthesized. This release step yields the polynucleotide insolution, which may then be separated from the solid support, e.g. byfiltration or other suitable methods. The release step is dependent uponhaving a support that is functionalized with a releasable moiety that,while inert under the conditions used in the synthesis cycle, providesfor the release of the synthesized polynucleotide under conditionsconducive for doing so.

The concept of a “safety catch linker” has been exploited widely. Theselinkers were originally developed by Kenner for peptide synthesis(Kenner et al. (1971) J. Chem. Soc. Chem. Commun. pp 636-37). They weredesigned to be cleaved in a two-stage process, where the first stepinvolves activation of a functional group on the linker, and the secondstep involves the actual cleavage of the linker. After the functionalgroup has been activated the cleavage step is more facile than it wouldhave been prior to activation. Kenner's safety catch linker is stable toboth acidic and basic conditions until the nitrogen is alkylated(activation), then cleaved by nucleophilic attack, for example withhydroxide or nucleophilic amine.

Another example of a safety catch linker, developed by Marshall andLiener (Marshall, D. L.; Liener, I. E., J. Org. Chem., 1970, 35,867-868), exploits the activation of a sulfide by oxidation to thesulfone. After activation with hydrogen peroxide, the linker is cleavedwith an amine nucleophile.

The concept of “safety catch linkers” has been further explored with avariety of type of activation methods prior to cleavage of the linker:most activation steps are performed through alkylation, oxidation, orneighboring group effects. However, these previously described processesare performed as two independent steps often requiring severalindependent reagents.

While there are examples of cleavable linkers in the literature, thereremains a need for novel cleavable linkers for polynucleotides, e.g.polynucleotides bound to a substrate.

SUMMARY OF THE INVENTION

In various embodiments of the invention, novel compositions having apolynucleotide bound to a substrate via a cleavable linker are provided,and methods of cleaving a polynucleotide from a substrate are provided.The cleavable linkers are cleavable under conditions that includecontact with an α-effect nucleophile.

Additional objects, advantages, and novel features of this inventionshall be set forth in part in the descriptions and examples that followand in part will become apparent to those skilled in the art uponexamination of the following specifications or may be learned by thepractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the materials and methodsparticularly pointed out in the appended claims.

DETAILED DESCRIPTION

Before the invention is described in detail, it is to be understood thatunless otherwise indicated this invention is not limited to particularmaterials, reagents, reaction materials, manufacturing processes, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting. It is also possible in the presentinvention that steps may be executed in different sequence where this islogically possible. However, the sequence described below is preferred.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an insoluble support” includes a plurality of insolublesupports. Similarly, reference to “a substituent”, as in a compoundsubstituted with “a substituent”, includes the possibility ofsubstitution with more than one substituent, wherein the substituentsmay be the same or different. In this specification and in the claimsthat follow, reference will be made to a number of terms that shall bedefined to have the following meanings unless a contrary intention isapparent:

A “nucleotide” refers to a sub-unit of a nucleic acid (whether DNA orRNA or analogue thereof) which includes a phosphate group, a sugar groupand a heterocyclic base, as well as analogs of such sub-units. A“nucleoside” references a nucleic acid subunit including a sugar groupand a heterocyclic base. A “nucleoside moiety” refers to a portion of amolecule having a sugar group and a heterocyclic base (as in anucleoside); the molecule of which the nucleoside moiety is a portionmay be, e.g. a polynucleotide, oligonucleotide, or nucleosidephosphoramidite. A “nucleobase” references the heterocyclic base of anucleoside or nucleotide. A “nucleotide monomer” refers to a moleculewhich is not incorporated in a larger oligo- or poly-nucleotide chainand which corresponds to a single nucleotide sub-unit; nucleotidemonomers may also have activating or protecting groups, if such groupsare necessary for the intended use of the nucleotide monomer. A“polynucleotide intermediate” references a molecule occurring betweensteps in chemical synthesis of a polynucleotide, where thepolynucleotide intermediate is subjected to further reactions to get theintended final product, e.g. a phosphite intermediate which is oxidizedto a phosphate in a later step in the synthesis, or a protectedpolynucleotide which is then deprotected.

As used herein, polynucleotides include single or multiple strandedconfigurations, where one or more of the strands may or may not becompletely aligned with another. The terms “polynucleotide” and“oligonucleotide” are generic to polydeoxynucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to anyother type of polynucleotide having nucleotide subunits that areN-glycosides of a purine or pyrimidine base, and to other polymers inwhich the conventional backbone has been replaced with a non-naturallyoccurring or synthetic backbone or in which one or more of theconventional bases has been replaced with a non-naturally occurring orsynthetic base. An “oligonucleotide” generally refers to a nucleotidemultimer of about 2 to 200 nucleotides in length, while a“polynucleotide” includes a nucleotide multimer having at least twonucleotides and up to several thousand (e.g. 5000, or 10,000)nucleotides in length. It will be appreciated that, as used herein, theterms “nucleoside”, “nucleoside moiety” and “nucleotide” will includethose moieties which contain not only the naturally occurring purine andpyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C), guanine(G), or uracil (U), but also modified purine and pyrimidine bases andother heterocyclic bases which have been modified (these moieties aresometimes referred to herein, collectively, as “purine and pyrimidinebases and analogs thereof”). Such modifications include, e.g.,methylated purines or pyrimidines, acylated purines or pyrimidines, andthe like, or the addition of a protecting group such as acetyl,difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, or the like. Thepurine or pyrimidine base may also be an analog of the foregoing;suitable analogs will be known to those skilled in the art and aredescribed in the pertinent texts and literature. Common analogs include,but are not limited to, 1-methyladenine, 2-methyladenine,N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-amninoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine.

The term “alkyl” as used herein, unless otherwise specified, refers to asaturated straight chain, branched or cyclic hydrocarbon group of 1 to24, typically 1-12, carbon atoms, such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl,neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl,2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term “lower alkyl” intendsan alkyl group of one to six carbon atoms, and includes, for example,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl. The term“cycloalkyl” refers to cyclic alkyl groups such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term “modified alkyl” refers to an alkyl group having from one totwenty-four carbon atoms, and further having additional groups, such asone or more linkages selected from ether-, thio-, amino-, phospho-,oxo-, ester-, and amido-, and/or being substituted with one or moreadditional groups including lower alkyl, aryl, alkoxy, thioalkyl,hydroxyl, amino, amido, sulfonyl, thio, mercapto, imino, halo, cyano,nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,silyl, silyloxy, and boronyl. The term “modified lower alkyl” refers toa group having from one to eight carbon atoms and further havingadditional groups, such as one or more linkages selected from ether-,thio-, amino-, phospho-, keto-, ester- and amido-, and/or beingsubstituted with one or more groups including lower alkyl; aryl, alkoxy,thioalkyl, hydroxyl, amino, amido, sulfonyl, thio, mercapto, imino,halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy,phosphoryl, silyl, silyloxy, and boronyl. The term “alkoxy” as usedherein refers to a substituent —O—R wherein R is alkyl as defined above.The term “lower alkoxy” refers to such a group wherein R is lower alkyl.The term “thioalkyl” as used herein refers to a substituent —S—R whereinR is alkyl as defined above.

The term “alkenyl” as used herein, unless otherwise specified, refers toa branched, unbranched or cyclic (e.g. in the case of C5 and C6)hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containingat least one double bond, such as ethenyl, vinyl, allyl, octenyl,decenyl, and the like. The term “lower alkenyl” intends an alkenyl groupof two to eight carbon atoms, and specifically includes vinyl and allyl.The term “cycloalkenyl” refers to cyclic alkenyl groups.

The term “alkynyl” as used herein, unless otherwise specified, refers toa branched or unbranched hydrocarbon group of 2 to 24, typically 2 to12, carbon atoms containing at least one triple bond, such asacetylenyl, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl,t-butynyl, octynyl, decynyl and the like. The term “lower alkynyl”intends an alkynyl group of two to eight carbon atoms, and includes, forexample, acetylenyl and propynyl, and the term “cycloalkynyl” refers tocyclic alkynyl groups.

The term “aryl” as used herein refers to an aromatic species containing1 to 5 aromatic rings, either fused or linked, and either unsubstitutedor substituted with 1 or more typically selected from the groupconsisting of lower alkyl, modified lower alkyl, aryl, aralkyl, loweralkoxy, thioalkyl, hydroxyl, thio, mercapto, amino, imino, halo, cyano,nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,silyl, silyloxy, and boronyl; and lower alkyl substituted with one ormore groups selected from lower alkyl, alkoxy, thioalkyl, hydroxyl thio,mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy,sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl.Typical aryl groups contain 1 to 3 fused aromatic rings, and moretypical aryl groups contain 1 aromatic ring or 2 fused aromatic rings.Aromatic groups herein may or may not be heterocyclic. The term“aralkyl” intends a moiety containing both alkyl and aryl species,typically containing less than about 24 carbon atoms, and more typicallyless than about 12 carbon atoms in the alkyl segment of the moiety, andtypically containing 1 to 5 aromatic rings. The term “aralkyl” willusually be used to refer to aryl-substituted alkyl groups. The term“aralkylene” will be used in a similar manner to refer to moietiescontaining both alkylene and aryl species, typically containing lessthan about 24 carbon atoms in the alkylene portion and 1 to 5 aromaticrings in the aryl portion, and typically aryl-substituted alkylene.Exemplary aralkyl groups have the structure —(CH2)j-Ar wherein j is aninteger in the range of 1 to 24, more typically 1 to 6, and Ar is amonocyclic aryl moiety.

The term “heterocyclic” refers to a five- or six-membered monocyclicstructure or to an eight- to eleven-membered bicyclic structure which iseither saturated or unsaturated. The heterocyclic groups herein may bealiphatic or aromatic. Each heterocyclic group consists of carbon atomsand from one to four heteroatoms selected from the group consisting ofnitrogen, oxygen and sulfur. As used herein, the term “nitrogenheteroatoms” includes any oxidized form of nitrogen and the quarteredform of nitrogen. The term “sulfur heteroatoms” includes any oxidizedform of sulfur. Examples of heterocyclic groups include purine,pyrimidine, piperidinyl, morpholinyl and pyrrolidinyl. “Heterocyclicbase” refers to any natural or non-natural heterocyclic moiety that canparticipate in base pairing or base stacking interaction on anoligonucleotide strand.

The term “halo” or “halogen” is used in its conventional sense to referto a chloro, bromo, fluoro or iodo substituent.

A “phospho” group includes a phosphodiester, phosphotriester, andH-phosphonate groups. In the case of either a phospho or phosphitegroup, a chemical moiety other than a substituted 5-membered furyl ringmay be attached to O of the phospho or phosphite group which linksbetween the furyl ring and the P atom.

By “protecting group” as used herein is meant a species which prevents aportion of a molecule from undergoing a specific chemical reaction, butwhich is removable from the molecule following completion of thatreaction, as taught for example in Greene, et al., “Protective Groups inOrganic Synthesis,” John Wiley and Sons, Second Edition, 1991. A“peroxyanion-labile linking group” is a linking group that releases alinked group when contacted with a solution containing peroxyanions.Similarly, a “peroxyanion-labile protecting group” is a protecting groupthat is removed from the corresponding protected group when contactedwith a solution containing peroxyanions. As used herein, “2′-Oprotecting groups” or “2′-hydroxyl protecting groups” are protectinggroups which protect the 2′-hydroxyl groups of the polynucleotide (e.g.bound to the 2′-O). As used herein, “phosphorus protecting group”(sometimes referenced as “phosphate protecting group”) references aprotecting group which protects a phosphorus group (e.g. is bound to aphosphorus group wherein the phosphorus group is attached to a sugarmoiety of, e.g. a nucleotide, a nucleoside phosphoramidite, apolynucleotide intermediate, or a polynucleotide). As used herein,“cleaving”, “cleavage”, “deprotecting”, “releasing”, or like terms whenused in reference to a protecting group refers to breaking a bond viawhich the protecting group is bound to the protected group, resulting inthe cleaved protecting group and the deprotected moiety (the moiety thatwas the protected group when bound to the protecting group).

The term “electron withdrawing” denotes the tendency of a substituent toattract valence electrons of the molecule of which it is a part, i.e.,an electron-withdrawing substituent is electronegative with respect toneighboring atoms. A quantification of the level of electron-withdrawingcapability is given by the Hammett sigma constant. This well knownconstant is described in many references, for instance, March, AdvancedOrganic Chemistry 251-59, McGraw Hill Book Company, New York, (1977).Exemplary electron-withdrawing groups include nitro, acyl, formyl,sulfonyl, trifluoromethyl, cyano, chloride, and the like.

The term “electron-donating” refers to the tendency of a substituent torepel valence electrons from neighboring atoms, i.e., the substituent isless electronegative with respect to neighboring atoms. Exemplaryelectron-donating groups include amino, methoxy, alkyl (including alkylhaving a linear or branched structure, alkyl having one to eightcarbons), cycloalkyl (including cycloalkyl having four to nine carbons),and the like.

The term “alpha effect,” as in an “alpha effect nucleophile” in adeprotection/oxidation agent, is used to refer to an enhancement ofnucleophilicity that is found when the atom adjacent a nucleophilic sitebears a lone pair of electrons. As the term is used herein, anucleophile is said to exhibit an “alpha effect” if it displays apositive deviation from a Bronsted-type nucleophilicity plot. Hoz et al.(1985) Israel J. Chem. 26:313. See also, Aubort et al. (1970) Chem.Comm. 1378; Brown et al. (1979) J. Chem. Soc. Chem. Comm.171; Buncel etal. (1982) J. Am. Chem. Soc. 104:4896; Edwards et al. (1962) J. Am.Chem. Soc. 84:16; Evanseck et al. (1987) J. Am. Chem Soc. 109:2349. Themagnitude of the alpha effect is dependent upon the electrophile whichis paired with the specific nucleophile. Mclsaac, Jr. et al. (1972), J.Org. Chem. 37:1037. Peroxy anions are example of nucleophiles whichexhibit strong alpha effects.

“Moiety” and “group” are used interchangeably herein to refer to aportion of a molecule, typically having a particular functional orstructural feature, e.g. a linking group (a portion of a moleculeconnecting two other portions of the molecule), or an ethyl moiety (aportion of a molecule with a structure closely related to ethane).

“Linkage” as used herein refers to a first moiety bonded to two othermoieties, wherein the two other moieties are linked via the firstmoiety. Typical linkages include ether (—O—), oxo (—C(O)—), amino(—NH—), amido (—N—C(O)—), thio (—S—), phosphate (—PO₄H—), ester(—O—C(O)—).

“Bound” may be used herein to indicate direct or indirect attachment. Inthe context of chemical structures, “bound” (or “bonded”) may refer tothe existence of a chemical bond directly joining two moieties orindirectly joining two moieties (e.g. via a linking group). The chemicalbond may be a covalent bond, an ionic bond, a coordination complex,hydrogen bonding, van der Waals interactions, or hydrophobic stacking,or may exhibit characteristics of multiple types of chemical bonds. Incertain instances, “bound” includes embodiments where the attachment isdirect and also embodiments where the attachment is indirect.

“Functionalized” references a process whereby a material is modified tohave a specific moiety bound to the material, e.g. a molecule orsubstrate is modified to have the specific moiety; the material (e.g.molecule or support) that has been so modified is referred to as afunctionalized material (e.g. functionalized molecule or functionalizedsupport).

The term “substituted” as used to describe chemical structures, groups,or moieties, refers to the structure, group, or moiety comprising one ormore substituents. As used herein, in cases in which a first group is“substituted with” a second group, the second group is attached to thefirst group whereby a moiety of the first group (typically a hydrogen)is replaced by the second group.

“Substituent” references a group that replaces another group in achemical structure. Typical substituents include nonhydrogen atoms (e.g.halogens), functional groups (such as, but not limited to amino,sulfhydryl, carbonyl, hydroxyl, alkoxy, carboxyl, silyl, silyloxy,phosphate and the like), hydrocarbyl groups, and hydrocarbyl groupssubstituted with one or more heteroatoms. Exemplary substituents includealkyl, lower alkyl, aryl, aralkyl, lower alkoxy, thioalkyl, hydroxyl,thio, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, and modified lower alkyl.

A “group” includes both substituted and unsubstituted forms. Typicalsubstituents include one or more lower alkyl, amino, imino, amido,alkylamino, arylamino, alkoxy, aryloxy, thio, alkylthio, arylthio,alkyl; aryl, thioalkyl, hydroxyl, mercapto, halo, cyano, nitro, nitroso,azido, carboxy, sulfide, sulfonyl, sulfoxy, phosphoryl, silyl, silyloxy,and boronyl optionally substituted on one or more available carbon atomswith a nonhydrocarbyl substituent such as cyano, nitro, halogen,hydroxyl, sulfonic acid, sulfate, phosphonic acid, phosphate, orphosphonate or the like. Any substituents are typically chosen so as notto substantially adversely affect reaction yield (for example, not lowerit by more than 20% (or 10%, or 5% or 1%) of the yield otherwiseobtained without a particular substituent or substituent combination).

As used herein, “dissociation constant”, e.g. an acid dissociationconstant, has its conventional definition as used in the chemical artsand references a characteristic property of a molecule having a tendencyto lose a hydrogen ion. The value of a dissociation constant mentionedherein is typically expressed as a negative log₁₀ value, i.e. a pKa (foran acid dissociation constant).

Hyphens, or dashes, are used at various points throughout thisspecification to indicate attachment, e.g. where two named groups areimmediately adjacent a dash in the text, this indicates the two namedgroups are attached to each other. Similarly, a series of named groupswith dashes between each of the named groups in the text indicates thenamed groups are attached to each other in the order shown. Also, asingle named group adjacent a dash in the text indicates the named groupis typically attached to some other, unnamed group. In some embodiments,the attachment indicated by a dash may be, e.g. a covalent bond betweenthe adjacent named groups. In some other embodiments, the dash mayindicate indirect attachment, i.e. with intervening groups between thenamed groups. At various points throughout the specification a group maybe set forth in the text with or without an adjacent dash, (e.g. amidoor amido-, further e.g. Lnk, Lnk- or -Lnk-) where the context indicatesthe group is intended to be (or has the potential to be) bound toanother group; in such cases, the identity of the group is denoted bythe group name (whether or not there is an adjacent dash in the text).Note that where context indicates, a single group may be attached tomore than one other group (e.g. where a linkage is intended, such aslinking groups).

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present, and, thus, thedescription includes structures wherein a non-hydrogen substituent ispresent and structures wherein a non-hydrogen substituent is notpresent. At various points herein, a moiety may be described as beingpresent zero or more times: this is equivalent to the moiety beingoptional and includes embodiments in which the moiety is present andembodiments in which the moiety is not present. If the optional moietyis not present (is present in the structure zero times), adjacent groupsdescribed as linked by the optional moiety are linked to each otherdirectly. Similarly, a moiety may be described as being either (1) agroup linking two adjacent groups, or (2) a bond linking the twoadjacent groups: this is equivalent to the moiety being optional andincludes embodiments in which the moiety is present and embodiments inwhich the moiety is not present. If the optional moiety is not present(is present in the structure zero times), adjacent groups described aslinked by the optional moiety are linked to each other directly.

We have now developed a set of conditions using peroxyanions that areeffective at performing both the activation and cleavage of certaintypes of cleavable linkers in a single step using a single reagent. Thisallows for more efficient chemical procedures as well as increasedflexibility due to the mildness of the cleavage conditions. Thus, invarious embodiments of the invention, novel compositions comprising apolynucleotide bound to a substrate via a cleavable linker are provided,and methods of cleaving a polynucleotide from a substrate are provided.

Accordingly, in certain embodiments of the present invention, acomposition is provided having a polynucleotide bound to a substrate viaa cleavable linker.

In some embodiments, the cleavable linker has the structure (I):

wherein:

-   -   R3, R4, R5, R6, R7, R10, and R11 are each independently selected        from H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl,        thio, mercapto, amino, amido, imino, halo, cyano, nitro,        nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,        silyl, silyloxy, boronyl or -Lnk-Sub, wherein Lnk is a linking        group and Sub denotes the site at which the substrate is        attached to the cleavable linker, provided that one and only one        of R3, R4, R5, R6, R7, R10, or R 11 is -Lnk-Sub; and    -   RPN denotes the site at which the polynucleotide is attached to        the cleavable linker.

In particular embodiments in which the cleavable linker has thestructure (I), R3, R4, R5, R6, R7, R10, and R11 are each independentlyselected from H, lower alkyl, modified lower alkyl, or -Lnk-Sub, whereinLnk is a linking group and Sub denotes the site at which the substrateis attached to the cleavable linker, provided that one and only one ofR3, R4, R5, R6, R7, R10, or R11 is -Lnk-Sub.

In particular embodiments in which the cleavable linker has thestructure (I), R3, R4, R6, R7, R10, and R11 are each independentlyselected from H or lower alkyl (e.g. from H, methyl, ethyl, n-propyl, orisopropyl); and R5 is -Lnk-Sub.

In some embodiments, the cleavable linker has the structure (II):

wherein:

-   -   R3, R4, R5, R6, R7, R10, and R11 are each independently selected        from H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl,        thio, mercapto, amino, amido, imino, halo, cyano, nitro,        nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,        silyl, silyloxy, boronyl or -Lnk-Sub, wherein Lnk is a linking        group and Sub denotes the site at which the substrate is        attached to the cleavable linker, provided that one and only one        of R3, R4, R5, R6, R7, R10, or R11 is -Lnk-Sub; and    -   RPN denotes the site at which the polynucleotide is attached to        the cleavable linker.

In particular embodiments in which the cleavable linker has thestructure (II), R3, R4, R5, R6, R7, R10, and R11 are each independentlyselected from H, lower alkyl, modified lower alkyl, or -Lnk-Sub, whereinLnk is a linking group and Sub denotes the site at which the substrateis attached to the cleavable linker, provided that one and only one ofR3, R4, R5, R6, R7, R10, or R11 is -Lnk-Sub.

In particular embodiments in which the cleavable linker has thestructure (II), R3, R4, R5, R6, R10, and R11 are each independentlyselected from H or lower alkyl (e.g. from H, methyl, ethyl, n-propyl, orisopropyl); and R7 is -Lnk-Sub.

In particular embodiments in which the cleavable linker has thestructure (II), R3, R4, R6, R7, R10, and R11 are each independentlyselected from H or lower alkyl (e.g. from H, methyl, ethyl, n-propyl, orisopropyl); and R5 is -Lnk-Sub.

In some embodiments, the cleavable linker has the structure (III):

wherein:

-   -   R4, R5, R6, and R7 are each independently selected from H, lower        alkyl, modified lower alkyl, thioalkyl, hydroxyl, thio,        mercapto, amino, amido, imino, halo, cyano, nitro, nitroso,        azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl,        silyloxy, boronyl, or -Lnk-Sub, wherein Lnk is a linking group        and Sub denotes the site at which the substrate is attached to        the cleavable linker, provided that one and only one of R4, R5,        R6, or R7 is -Lnk-Sub;    -   R13 is selected from lower alkyl, modified lower alkyl, alkyl,        modified alkyl, or aryl; and    -   RPN denotes the site at which the polynucleotide is attached to        the cleavable linker.

In particular embodiments in which the cleavable linker has thestructure (III), R4, R5, R6, and R7 are each independently selected fromH, lower alkyl, modified lower alkyl, or -Lnk-Sub, wherein Lnk is alinking group and Sub denotes the site at which the substrate isattached to the cleavable linker, provided that one and only one of R4,R5, R6, or R7 is -Lnk-Sub.

In particular embodiments in which the cleavable linker has thestructure (III), R4, R6, R7, R10, and R11 are each independentlyselected from H or lower alkyl (e.g. from H, methyl, ethyl, n-propyl, orisopropyl); R5 is -Lnk-Sub; and R13 is lower alkyl (e.g. selected fromH, methyl, ethyl, n-propyl, or isopropyl).

In some embodiments, the cleavable linker has the structure (IV):

wherein:

-   -   R4, R5, R6, R7, R10, and R11 are each independently selected        from H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl,        thio, mercapto, amino, amido, imino, halo, cyano, nitro,        nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,        silyl, silyloxy, boronyl or -Lnk-Sub, wherein Lnk is a linking        group and Sub denotes the site at which the substrate is        attached to the cleavable linker, provided that one and only one        of R4, R5, R6, R7, R10, or R11 is -Lnk-Sub;    -   R13 is selected from lower alkyl, modified lower alkyl, alkyl,        or modified alkyl, or aryl; and    -   RPN denotes the site at which the polynucleotide is attached to        the cleavable linker;

In particular embodiments in which the cleavable linker has thestructure (IV), R4, R5, R6, R7, R10, and R11 are each independentlyselected from H, lower alkyl, modified lower alkyl, or -Lnk-Sub, whereinLnk is a linking group and Sub denotes the site at which the substrateis attached to the cleavable linker, provided that one and only one ofR4, R5, R6, R7, R10, or R11 is -Lnk-Sub;

In particular embodiments in which the cleavable linker has thestructure (IV), R4, R6, R7, R10, and R11 are each independently selectedfrom H or lower alkyl (e.g. from H, methyl, ethyl, n-propyl, orisopropyl); R5 is -Lnk-Sub; and R13 is lower alkyl (e.g. selected fromH, methyl, ethyl, n-propyl, or isopropyl).

In some embodiments, the cleavable linker has the structure (V):

wherein:

-   -   R4, R5, R6, R7, R10, and R11 are each independently selected        from H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl,        thio, mercapto, amino, amido, imino, halo, cyano, nitro,        nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl,        silyl, silyloxy, boronyl, or -Lnk-Sub, wherein Lnk is a linking        group and Sub denotes the site at which the substrate is        attached to the cleavable linker, provided that one and only one        of R4, R5, R6, R7, R10, or R11 is -Lnk-Sub;    -   R13 is selected from lower alkyl, modified lower alkyl, alkyl,        or modified alkyl, or aryl; and    -   RPN denotes the site at which the polynucleotide is attached to        the cleavable linker.

In particular embodiments in which the cleavable linker has thestructure (V), R4, R5, R6, R7, R10, and R11 are each independentlyselected from H, lower alkyl, modified lower alkyl, or -Lnk-Sub, whereinLnk is a linking group and Sub denotes the site at which the substrateis attached to the cleavable linker, provided that one and only one ofR4, R5, R6, R7, R10, or R11 is -Lnk-Sub.

In particular embodiments in which the cleavable linker has thestructure (V), R4, R6, R7, R10, and R11 are each independently selectedfrom H or lower alkyl (e.g. from H, methyl, ethyl, n-propyl, orisopropyl); R5 is -Lnk-Sub; and R13 is lower alkyl (e.g. selected fromH, methyl, ethyl, n-propyl, or isopropyl).

The polynucleotide attached to the substrate via the cleavable linkermay be any polynucleotide, for example DNA, RNA, a polynucleotideanalog, a modified polynucleotide, a polynucleotide having protectinggroups (e.g. protecting groups bound to the amine groups of nucleobases,protecting groups bound to the phosphate groups of the polynucleotide,protecting groups which protect hydroxyl groups of the polynucleotide(e.g. bound to the 2′-O), or other protecting groups). Thepolynucleotide may be synthesized in situ (e.g. synthesized onenucleotide at a time using polynucleotide synthesis schemes well knownin the art) or may be separately synthesized and then attached to thesubstrate via the cleavable linker. For example, a modified cleavablelinker moiety is bound to the substrate, wherein the modified cleavablelinker moiety has a protected nucleotide moiety bound to a cleavablelinker moiety as described herein. The protected nucleotide moiety isthen deprotected, and the deprotected nucleotide moiety serves as a siteto either start in situ synthesis of a full length polynucleotide or asa site for attachment of an already synthesized polynucleotide. Othermethods of providing the polynucleotide attached to the substrate viathe cleavable linker are possible and may be employed in accordance withthe present invention. The polynucleotide may generally be attached tothe cleavable linker via any available site of the polynucleotide, e.g.at the 2′-O, the 3′-O, the 5′-O, an amino group of a nucleobase, or anyother site, given that the available site provides a resulting structurethat is cleavable upon contacting the polynucleotide-bound substratewith the a-effect nucleophile. Typically, the polynucleotide is attachedto the cleavable linker at the 2′-O or the 3′-O, less typically at the5′-O or at an amino group of a nucleobase.

In particular embodiments, the polynucleotide has a 2′-hydroxylprotecting group and at least one additional protecting group selectedfrom a nucleobase protecting group and a phosphorus protecting group,wherein said 2′-hydroxyl protecting group is characterized as stableunder conditions which include an a-effect nucleophile; and wherein saidat least one additional protecting group is characterized as labileunder conditions which include an α-effect nucleophile.

In particular embodiments, the polynucleotide has a 2′-hydroxylprotecting group, a phosphorus protecting group, and a nucleobaseprotecting group, wherein the 2′-hydroxyl protecting group and phosphateprotecting group are characterized as stable under conditions whichinclude an a-effect nucleophile; and wherein the nucleobase protectinggroup is characterized as labile under conditions which include anα-effect nucleophile.

Referring to structures (I), (II), (III), (IV), (V), the linking group-Lnk- is selected from (1) a linking group linking the substrate and thecleavable linker; or (2) a covalent bond between the substrate and thecleavable linker (e.g. the cleavable linker is directly bound to thesubstrate). In particular embodiments, the linking group -Lnk- may beany appropriate linking group via which the substrate is attached to thecleavable linker. The linking group -Lnk- is typically selected from (1)a lower alkyl group; (2) a modified lower alkyl group in which one ormore linkages selected from ether-, thio-, amino-, oxo-, ester-, andamido- is present; (3) a modified lower alkyl substituted with one ormore groups including lower alkyl; aryl, aralkyl, alkoxyl, thioalkyl,hydroxyl, amino, amido, sulfonyl, halo; or (4) a modified lower alkylsubstituted with one or more groups including lower alkyl; alkoxyl,thioalkyl, hydroxyl, amino, amido, sulfonyl, halo, and in which one ormore linkages selected from ether-, thio-, amino-, oxo-, ester-, andamido- is present. The linking group -Lnk- may be bonded to thesubstrate at any position of the linking group -Lnk- available to bindto the substrate. Similarly, the linking group -Lnk- may be bonded tothe adjacent cleavable linker at any position of the linking group -Lnk-available to bind to the adjacent cleavable linker. In certainembodiments, the linking group -Lnk- is a single methylene group, e.g.-CH2-, or may be an alkyl group or modified alkyl group up to about 24carbons long (and which may be straight-chain or branched-chain). Incertain such embodiments, one or more linkages selected from ether-,oxo-, thio-, and amino- is present in the straight-or branched chainmodified alkyl group. In an embodiment, the linking group -Lnk-comprises optionally substituted ethoxy, propoxy, or butoxy groups (i.e.may include the structure —{(CH2)m-O}n-, wherein m is a integer selectedfrom 2, 3, 4, and n is a integer selected from 1, 2, 3, 4, 5, 6). In anembodiment, the linking group -Lnk- has the structure—(CH2)m-Lkg-(CH2)n-, wherein m and n are integers independently selectedfrom the range of 1 to about 12, e.g. from the range of 2 to about 8,and Lkg is a linkage selected from ether-, thio-, amino-, oxo-, ester-,and amido-.

In particular embodiments, the linking group -Lnk- has a first terminalsite and a second terminal site. In such embodiments, the linking group-Lnk- is bound to the substrate at the first terminal site, and thelinking group -Lnk- is bound to the cleavable linker at the secondterminal site. The first and second terminal sites will depend on thedesign of the linking group taking into consideration, for example, themethod used to attach the cleavable linker to the substrate.

The cleavable linker can be attached to a suitable substrate that mayhave a variety of forms and compositions. The substrate may derive fromnaturally occurring materials, naturally occurring materials that havebeen synthetically modified, or synthetic materials. Examples ofsuitable support materials include, but are not limited to,nitrocellulose, glasses, silicas, teflons, and metals (e.g., gold,platinum, and the like). Suitable materials also include polymericmaterials, including plastics (for example, polytetrafluoroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike), polysaccharides such as agarose (e.g., that availablecommercially as Sepharose®, from Pharmacia) and dextran (e.g., thoseavailable commercially under the tradenames Sephadex® and Sephacyl®,also from Pharmacia), polyacrylamides, polystyrenes, polyvinyl alcohols,copolymers of hydroxyethyl methacrylate and methyl methacrylate, and thelike.

The cleavable linker may be bound directly to the substrate (e.g. to thesurface of the substrate, e.g. to a functional group on the surface) orindirectly bound to the substrate, e.g. via one or more intermediatemoieties (e.g. linking groups) and/or surface modification layer on thesubstrate. The nature of the site on the substrate to which thecleavable linker is attached (e.g. directly or via a linking group) isnot essential to the present invention, as any known coupling chemistrycompatible with the sensor substrate (i.e. which doesn't result insignificant degradation of the sensor substrate) may be used to coupleto the cleavable linker. As such, various strategies of coupling thecleavable linker to substrates using functional groups on the substratesare known in the art and may be employed advantageously in the disclosedmethods. Typical strategies require a complementary reactive group onthe cleavable linker or are selected based on moieties already presenton the cleavable linker (e.g. amino groups, hydroxyl groups, or otherfunctional groups), for example an active group on the substrate that iscapable of reacting with a corresponding reactive group attached to thelinker to result in the linker covalently bound to the substrate.

Accordingly, in certain embodiments of the present invention, a methodis provided wherein the method includes: contacting a polynucleotidebound to a substrate via a cleavable linker with a solution comprisingan α-effect nucleophile to result in cleavage of the polynucleotide fromthe substrate; wherein the cleavable linker has a structure selectedfrom structures (I), (II), (III), (IV), or (V), as described above.

As mentioned above, embodiments of the present disclosure includemethods for cleaving a polynucleotide from a substrate, wherein thepolynucleotide is bound to a cleavable linker such as those describedherein. In particular embodiments, the method includes contacting thepolynucleotide bound to the substrate via the cleavable linker with asolution of an α-effect nucleophile (e.g., a peroxyanion solution),where the α-effect nucleophile has a pKa of about 4 to 13. In addition,the solution is at a pH of about 6 to 11.

In particular embodiments, a polynucleotide bound to a substrate via acleavable linker is contacted with a solution of peroxyanions to resultin cleavage of the polynucleotide from the substrate, wherein theperoxyanions have a pKa within the range 4-12, at neutral to mildlybasic pH (e.g. the pH typically is in the range from about 6 to about11).

In typical embodiments, the conditions employed for deprotection includecontacting the polynucleotide with the solution of the α-effectnucleophile for time sufficient to result in cleavage of the phophorusprotecting group. Typical times (duration) for the cleavage reactionrange from about 15 minutes to about 24 hour, although times outsidethis range may be used. Typically the duration of the contacting is inthe range from about 30 minutes to about 16 hours, e.g. from about 45minutes to about 12 hours, from about 1 hour to about 8 hours, or fromabout 1 hour to about 4 hours.

One advantage of using a neutral to mildly basic (e.g. pH in the rangefrom about 6 to about 11) solution including an α-effect nucleophile isthat the solution including an α-effect nucleophile is compatible withstandard phosphoramidite methods for polynucleotide synthesis. Further,the polynucleotides released from the substrate by cleavage of thecleavable linker are stable and show little or no degradation for anextended period of time when stored in the solution including theα-effect nucleophile.

In general, the solution including the α-effect nucleophile can be apredominantly buffered aqueous solution or buffered aqueous/organicsolution. Under these conditions it is convenient and cost effective torecover the released polynucleotide from the mixture of releasedpolynucleotide and solution of α-effect nucleophile by simpleprecipitation of the desired polynucleotides directly from the mixtureby addition of ethanol to the mixture. Under these conditions, thepolynucleotide is pelleted to the bottom of a centrifuge tube and thesupernatant containing the α-effect nucleophile removed by simplypouring off the supernatant and rinsing the pellet with fresh ethanol.The released polynucleotide is then isolated by resuspending in atypical buffer for chromatographic purification or direct usage in thebiological experiment of interest. Because of the nature of mostα-effect nucleophiles, removal from the desired released polynucleotideproducts is easy, quick, and effective using the ethanol precipitationmethod. Any other methods of recovering the polynucleotides may beemployed, such as using Micro Bio-Spin™ chromatography columns (BioRad,Hercules, Calif.) for cleanup and purification of polynucleotides (usedaccording to product insert instructions).

The solution including the α-effect nucleophile typically may have a pHin the range of about 4 to 11, about 5 to 11, about 6 to 11, about 7 to11, about 8 to 11, about 4 to 10, about 5 to 10, about 6 to 10, about 7to 10, or about 8 to 10. In particular embodiments the solution has a pHof about 7 to 10. It should also be noted that the pH is dependent, atleast in part, upon the α-effect nucleophile in the solution and theprotecting groups on the polynucleotide. Appropriate adjustments to thepH can be made to the solution to accommodate the α-effect nucleophile.

The α-effect nucleophiles can include, but are not limited to,peroxyanions, hydroxylamine derivatives, hydroximic acid and derivativesthererof, hydroxamic acid and derivatives thereof, carbazide andsemicarbazides and derivatives thereof. The α-effect nucleophiles caninclude compounds such as, but not limited to, hydrogen peroxide,peracids, perboric acid salts, alkylperoxides, hydrogen peroxide salts,hydroperoxides, butylhydroperoxide, benzylhydroperoxide,phenylhydroperoxide, cumene hydroperoxide, performic acid, peraceticacid, perbenzoic acid and substituted perbenzoic acids such aschloroperbenzoic acid, perbutyric acid, tertiary-butylperoxybenzoicacid, decanediperoxoic acid, other similar compounds, and correspondingsalts, and combinations thereof. Hydrogen peroxide, salts of hydrogenperoxide and mixtures of hydrogen peroxide and performic acid areespecially useful. Hydrogen peroxide, whose pKa is around 11, isparticularly useful in solutions above pH 9.0. Below pH 9.0 there is nosignificant concentration of peroxyanion to work as an effectivenucleophile. Below pH 9.0 it is especially useful to use mixtures ofhydrogen peroxide and peracids. These peracids can be preformed andadded to the solution or they can be formed in situ by the reaction ofhydrogen peroxide and the carboxylic acid or carboxylic acid salt. Anexample is that an equal molar mixture of hydrogen peroxide and sodiumformate can be used at pH conditions below 9.0 as an effective α-effectnucleophile solution where hydrogen peroxide alone is not provide a highconcentration of α-effect nucleophiles. The utility of peracids tends tobe dependent upon the pKa of the acid and size of molecule: the higherthe pKa of the acid the more useful as a peroxyanion solution, thelarger the size of the molecule the less useful. Typically the pKa ofthe peracid is lower than the pH of the desired peroxyanion solution.

The α-effect nucleophiles typically used in these reactions aretypically strong oxidants, therefore one should limit the concentrationof the reagent in the solution in order to avoid oxidative side productswhere undesired. The α-effect nucleophiles are typically less than 30%weight/vol of the solution, more typically between 0.1% and 10%weight/vol of the solution and most typically 3% to 7% weight/vol of thesolution. The typical 3% solution of hydrogen peroxide is about 1 molarhydrogen peroxide. A solution of between 1 molar and 2 molar hydrogenperoxide is typically useful. A typical solution of hydrogen peroxideand performic acid is an equal molar mixture of hydrogen peroxide andperformic acid, both in the range of 1 to 2 molar. An example of an insitu prepared solution of performic acid is 2 molar hydrogen peroxideand 2 molar sodium formate buffered at pH 8.5.

In typical embodiments, the α-effect nucleophile is characterized ashaving a pKa in the range from about 4 to 13, about 4 to 12, about 4 to11, about 5 to 13, about 5 to 12, about 5 to 11, about 6 to 13, about 6to 12, about 6 to 11, about 7 to 13, about 7 to 12, or about 7 to 11.

It should also be noted that the dissociation constant is a physicalconstant that is characteristic of the specific α-effect nucleophile.Chemical substitution and solvent conditions can be used to raise orlower the effective dissociation constant and therefore specificallyoptimize the conditions under which the cleavage of the cleavable linkeris performed (to result in release of the polynucleotide from thesubstrate, and, optionally, deprotection of groups protected byperoxyanion-labile protecting groups). Appropriate selection of theα-effect nucleophile should be made considering the other conditions ofthe method and the protecting groups of the polynucleotide. In addition,mixtures of carboxylic acids and hydroperoxides can be used to formsalts of peracids in situ.

As an example a solution of hydrogen peroxide can be used with asolution of formic acid at pH conditions below 9.0. At pH conditionsless than 9.0 hydrogen peroxide is not significantly ionized due to itsionization constant of around 11. At pH 7.0 only about 0.01% of thehydrogen peroxide is in the ionized form of the α-effect nucleophile.However, the hydrogen peroxide can react in situ with the formic acid toform performic acid in a stable equilibrium. At pH 7.0 the performicacid is significantly in the ionized form and is an active α-effectnucleophile. The advantage of such an approach is that solutions ofperformic acid tend to degrade rapidly and stabilizers need to be added.The equilibrium that is formed between the hydrogen peroxide solutionsand the formic acid helps stabilize the performic acid such that it canbe used to completely cleave the polynucleotides from the substratesprior to degrading. Performic acid is especially useful in a bufferedmixture of hydrogen peroxide at pH 8.5 because the pKa of performic acidis approximately 7.1. Peracetic acid is useful at pH 8.5 but less usefulthan performic acid because the pKa of peracetic acid is approximately8.2. At pH 8.5 peracetic acid is only about 50% anionic whereas at pH8.5 performic acid is more than 90% anionic.

In general, the pKa for the hydroperoxides is about 8 to 13. The pKa forhydrogen peroxide is quoted to be about 10 to 12 depending upon themethod of analysis and solvent conditions. The pKa for thealkylperoxides is about 8 to 14. The pKa for the peracids is about 3 to9. In some embodiments in which the peroxyanion is hydroperoxide, thesolution is at pH of about 9 to 11, e.g. at a pH of about 9 to about 10.In certain embodiments in which the peroxyanion is an alkylperoxide, thesolution is at pH of about 8 to 11. In embodiments where the peroxyanionis a peracid, the solution is at pH of about 6 to 9. In addition, theperacid typically has a pKa of about 4 to 10.

In addition, the aqueous buffer solution usually includes a buffer, suchas, but not limited to, tris(hydroxymethyl)aminomethane,aminomethylpropanol, citric acid, N,N′-Bis(2-hydroxyethyl)glycine,2-[Bis(2-hydroxyethyl)amino]-2-(hydroxy-methyl)-1,3-propanediol,2-(Cyclohexylamino)ethane-2-sulfonic acid,N-2-Hydroxyethyl)piperazine-N′-2-ethane sulfonic acid,N-(2-Hydroxyethyl)piperazine-N′-3-propane sulfonic acid,Morpholinoethane sulfonic acid, Morpholinopropane sulfonic acid,Piperazine-N,N′-bis(2-ethane sulfonic acid),N-Tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid,N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid, N-Tris(hydroxymethyl) methylglycine, and combinations thereof.

One significant potential advantage for cleaving the polynucleotide froma substrate according to the present methods is that the (α-effectnucleophile solution can be exploited to remove a variety ofperoxyanion-labile protecting groups at the same time and under the sameconditions that are used to cleave the polynucleotide from thesubstrate. Thus, cleavage of the polynucleotide from the substrate anddeprotection of groups protected with peroxyanion-labile protectinggroups may be reduced to a single step in which the cleavage anddeprotection occur at essentially the same time in the same reactionmixture. These advantages become even more significant if they are usedwith the protecting groups described in the applications cited herein toDellinger et al. that were filed on the same day as the presentapplication; such protecting groups specifically provide for rapiddeprotection under the oxidative, nucleophilic conditions at neutral tomildly basic pH.

Particularly contemplated is the use of the cleavable linkers describedherein in conjunction with peroxyanion-labile protecting groups attachedto the polynucleotide. The peroxyanion-labile protecting groups may beattached, e.g. at the 2′-position of the nucleoside sugar of theindividual nucleotide subunits, at the exocyclic amine groups of theheterocyclic bases of the polynucleotide, at the imine groups of theheterocyclic bases of the polynucleotide, and/or at the phosphate groupsof the backbone of the polynucleotide. In certain such embodiments,contacting the polynucleotide with solution including an α-effectnucleophile results in concurrent cleavage of the polynucleotide fromthe substrate and deprotection of the polynucleotide, e.g. at the2′-position of the nucleoside sugar, at the exocyclic amine groups, atthe imine groups of the heterocyclic bases, and/or at the phosphategroups.

For example, in particular embodiments a polynucleotide bound to asubstrate via a cleavable linker as described herein hasperoxyanion-labile protecting groups on, e.g. the exocyclic aminegroups. In some such embodiments, contacting the polynucleotide withsolution including an α-effect nucleophile results in concurrentcleavage of the polynucleotide from the substrate and deprotection ofthe exocyclic amine groups. As another example, in particularembodiments a polynucleotide bound to a substrate via a cleavable linkeras described herein has peroxyanion-labile protecting groups on, e.g.the 2′ position of the nucleoside sugar. In certain such embodiments,contacting the polynucleotide with a solution including an α-effectnucleophile results in concurrent cleavage of the polynucleotide fromthe substrate and deprotection of the 2′ position of the nucleosidesugar (e.g. resulting in a deprotected 2′-hydroxyl group). In a furtherexample, a polynucleotide bound to a substrate via a cleavable linkerhas peroxyanion-labile protecting groups on, e.g. the 2′ position of thenucleoside sugar and the exocyclic amine groups. In certain suchembodiments, contacting the polynucleotide with a solution including anα-effect nucleophile results in concurrent cleavage of thepolynucleotide from the substrate and deprotection of the 2′ position ofthe nucleoside sugar and the exocyclic amine groups.

Structure (VII) serves to illustrate a portion of a polynucleotide boundto a substrate, and illustrates that there are several sites of thepolynucleotide which may have protecting groups bound thereto, includingphosphorus protecting groups (designated R in structure (VII), andsometimes referenced herein as “phosphate protecting groups”),nucleobase protecting groups (designated R″ in structure (VII)); and2′-hydroxyl protecting groups (designated R′ in structure (VII), andsometimes referenced herein as 2′-O protecting groups). Note thatstructure (VII) only depicts two nucleotide subunits, but that typicallythere will be many more nucleotide subunits in the polynucleotide havingthe same general structure as the nucleotide subunits depicted instructure (VII). In structure (VII), B represents a nucleobase. It iscontemplated that, in particular embodiments, the protecting groups(i.e. one or more of R, R′, and/or R″) may be labile under the sameconditions that result in cleavage of the cleavable linkers (CLG instructure (VII)) described herein.

In particular embodiments the polynucleotide has a plurality ofphosphate groups wherein each phosphate group is bound to a phosphateprotecting group. In certain such embodiments, the phosphate protectinggroup is labile under the same conditions as the cleavable linker (e.g.the phosphate protecting group is peroxyanion-labile). Thus, thecleavable linker may be cleaved and the phosphate groups may undergodeprotection concurrently upon being contacted with a solutioncomprising an α-effect nucleophile. Any phosphate protecting group knownin the art of polynucleotide synthesis that is labile under conditionsof cleavage of the cleavable linker may be used. Examples of suchphosphate protecting groups are described in a copending U.S. patentapplication Ser. No. 11/338,339 filed on the same day as the instantapplication by Dellinger et al. entitled “Phosphorus Protecting Groups”.

Thus, in particular embodiments, the present invention provides for amethod that includes: contacting a polynucleotide bound to a substratevia a cleavable linker with a solution comprising an α-effectnucleophile; wherein the polynucleotide has a plurality of phosphategroups; wherein each phosphate group of the plurality of phosphategroups has a phosphate protecting group bound thereto, said phosphateprotecting group characterized as being labile upon exposure to theα-effect nucleophile, said contacting resulting in concurrent cleavageof the polynucleotide from the substrate and deprotection of eachphosphate group of the plurality of phosphate groups.

In particular embodiments the polynucleotide has a plurality ofnucleobases, wherein each nucleobase is bound to a nucleobase protectinggroup. In certain such embodiments, the nucleobase protecting group islabile under the same conditions as the cleavable linker (e.g. thenucleobase protecting group is peroxyanion-labile). Thus, the cleavablelinker may be cleaved and the nucleobases may undergo deprotectionconcurrently upon being contacted with a solution comprising an α-effectnucleophile. Any nucleobase protecting group known in the art ofpolynucleotide synthesis that is labile under conditions of cleavage ofthe cleavable linker may be used. Examples of such nucleobase protectinggroups are described in a copending application Ser. No. 11/387,388filed on the same day as the instant application by Dellinger et al.entitled “Monomer Compositions for the Synthesis of Polynucleotides,Methods of Synthesis, and Methods of Deprotection”.

Thus, in particular embodiments, the present invention provides for amethod that includes: contacting a polynucleotide bound to a substratevia a cleavable linker with a solution comprising an α-effectnucleophile; wherein the polynucleotide has a plurality of nucleobases;wherein each nucleobase of the plurality of nucleobases has a nucleobaseprotecting group bound thereto, said nucleobase protecting groupcharacterized as being labile upon exposure to the α-effect nucleophile,said contacting resulting in concurrent cleavage of the polynucleotidefrom the substrate and deprotection of each nucleobase of the pluralityof nucleobases.

In particular embodiments the polynucleotide has a plurality of 2′-Ogroups wherein each 2′-O group is bound to a 2′-O protecting group (i.e.a 2′-hydroxyl protecting group). In certain such embodiments, the 2′-Oprotecting group is labile under the same conditions as the cleavablelinker (e.g. the 2′-O protecting group is peroxyanion-labile). Thus, thecleavable linker may be cleaved and the 2′-O groups may undergodeprotection concurrently upon being contacted with a solutioncomprising an α-effect nucleophile. Any 2′-O protecting group known inthe art of polynucleotide synthesis that is labile under conditions ofcleavage of the cleavable linker may be used. Examples of such 2′-Oprotecting groups are described in a copending application Ser. No.11/388,112 filed on the same day as the instant application by Dellingeret al. entitled “Monomer Compositions for the Synthesis ofPolynucleotides, Methods of Synthesis, and Methods of Deprotection”.

Thus, in particular embodiments, the present invention provides for amethod that includes: contacting a polynucleotide bound to a substratevia a cleavable linker with a solution comprising an α-effectnucleophile; wherein the polynucleotide has a plurality of 2′-O groups;wherein each 2′-O group of the plurality of 2′-O groups has a 2′-Oprotecting group bound thereto, said 2′-O protecting group characterizedas being labile upon exposure to the α-effect nucleophile, saidcontacting resulting in concurrent cleavage of the polynucleotide fromthe substrate and deprotection of each 2′-O group of the plurality of2′-O groups.

Furthermore, in certain embodiments, the polynucleotide includes aplurality of phosphate groups, a plurality of nucleobases, and aplurality of 2′-O groups. In some embodiments, the polynucleotide alsoincludes one or more (e.g. two or more, e.g. all three) types ofprotecting groups selected from phosphate protecting groups, nucleobaseprotecting groups, or 2′-O protecting groups. Each protecting group isbound to a corresponding site of the polynucleotide (i.e. phosphateprotecting groups are bound to phosphate groups, nucleobase protectinggroups are bound to nucleobases, and 2′-O protecting groups are bound to2′-O groups). In certain embodiments, the method provides fordeprotection of the polynucleotide and concurrent cleavage of thepolynucleotide from the substrate. The concurrent deprotection andcleavage may include deprotection of: 1) the phosphate groups 2) thenucleobases, 3) the 2′-O groups; 4) phosphate groups and thenucleobases, 5) the nucleobases and the 2′-O groups, 6) phosphate groupsand the 2′-O groups; or 7) the phosphate groups, the nucleobases, andthe 2′-O groups.

In certain embodiments, the polynucleotide has a 2′-hydroxyl protectinggroup and at least one additional protecting group selected from anucleobase protecting group and/or a phosphorus protecting group,wherein the 2′-hydroxyl protecting group is stable under conditionswhich include an α-effect nucleophile. In this regard, “stable” meansthat the protecting group is not susceptible to being cleaved (removedfrom the protected group) upon being contacted with an α-effectnucleophile. However, in these embodiments, the nucleobase protectinggroup and/or a phosphorus protecting group are labile under conditionswhich include an α-effect nucleophile; thus, the contacting with thesolution of α-effect nucleophile results in concurrent cleavage of thepolynucleotide from the substrate and cleavage of said nucleobaseprotecting groups and/or a phosphorus protecting groups. In some suchembodiments, the method further provides for cleaving the 2′hydroxylprotecting group under conditions sufficient to result in cleavage ofthe 2′hydroxyl protecting group, wherein said conditions do not includeα-effect nucleophile. The cleaving of the 2′hydroxyl protecting group insuch embodiments may be either before or after the contacting withα-effect nucleophile (i.e. before or after the concurrent cleavage ofthe cleavable linker and nucleobase protecting group and/or a phosphorusprotecting group).

In certain embodiments, the polynucleotide has a 2′-hydroxyl protectinggroup, a phosphorus protecting group, and a nucleobase protecting group;wherein the 2′-hydroxyl protecting group and phosphorus protecting groupare stable under conditions which include an α-effect nucleophile. Inthese embodiments, the nucleobase protecting group is labile underconditions which include an α-effect nucleophile; thus the contactingwith the solution of α-effect nucleophile results in concurrent cleavageof the polynucleotide from the substrate and cleavage of said nucleobaseprotecting group. In some such embodiments, the method further providesfor cleaving the 2′-hydroxyl protecting group and/or the phosphorusprotecting group under conditions sufficient to result in cleavage ofthe 2′-hydroxyl protecting group and/or the phosphorus protecting group,wherein said conditions do not include α-effect nucleophile. Thecleaving of the 2′-hydroxyl protecting group and/or the phosphorusprotecting group in such embodiments may be before or after thecontacting with α-effect nucleophile (i.e. before or after theconcurrent cleavage of the cleavable linker and nucleobase protectinggroup)

FURTHER EXAMPLES

In an embodiment, a solution including a peroxyanion is used to performoxidation and nucleophilic cleavage of an oxidizable cleavable linker ina single step. An example of this cleavage is shown below, wherein R isthe polynucleotide linked to the solid support via the oxidizablecleavable linker:

The carbonate group shown in the left of this scheme is stabile underacidic, neutral, and mildly basic conditions. The electron donatingeffect of the thioether in the para position to the carbonate addsstability to the linkage. However, the thiol ether can be oxidized tothe sulfoxide and further to the sulfone using a dilute solution ofhydrogen peroxide in a buffered aqueous solution at pH 8.0 (mildlybasic), thus rendering the carbonate much more susceptible to cleavageby nucleophiles. The cleavage then occurs by the action of theperoxyanion as a strong nucleophile; in this process both the activationand cleavage of the linker occur in a one-pot reaction using a singlereagent.

In another embodiment, cleavable linkers that require activation viaparticipation of a neighboring group can also be cleaved in one stepunder mild pH conditions using peroxyanions. An example of this type oflinker is shown below, wherein R is the polynucleotide linked to thesolid support via the oxidizable cleavable linker:

The neighboring group effect of the phenol can significantly affect therate of cleavage of the ester attached to R by participating in thestabilization of the tetrahedral cleavage intermediate. Therefore highlystable esters are cleaved more rapidly after the release of theneighboring phenol and its acidic proton.

In yet another embodiment, oxidation and neighboring group participationcan be combined. One significant advantage in using peroxyanions is theability to affect the acidity of the phenol using the oxidizingcapability of peroxyanions. An example of this is the use of athiomethyl group on the ring that, once oxidized, increases the rate ofphenol formation, and results in a more acidic neighboring group phenol.Such an example is shown below, wherein R is the polynucleotide linkedto the solid support via the oxidizable cleavable linker:

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,biochemistry, molecular biology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

The following description is put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to perform the methods and use the compositions disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, percents are wt./wt., temperature is in ° C. andpressure is at or near atmospheric. Standard temperature and pressureare defined as 20 ° C. and 1 atmosphere.

A synthesis of reagents used in certain embodiments of the presentinvention is now described. It will be readily apparent that thereactions described herein may be altered, e.g. by using modifiedstarting materials to provide correspondingly modified products, andthat such alteration is within ordinary skill in the art. Given thedisclosure herein, one of ordinary skill will be able to practicevariations that are encompassed by the description herein without undueexperimentation.

Another example of a composition useful in certain embodiments having acleavable linker is represented below.

In this example, the linker is connected to the nucleoside through acarbonate group which is stable under moderate basic conditions but thatbecomes much more sensitive to hydrolysis when the thioether is oxidizedto a sulfone. Here again, the sulfone makes the carbon on the carbonategroup much more electrophilic, by its electron withdrawing effect.

Preparation of this Compound:

4-Mercaptophenol (5 g) was dissolved in abs. DMF (dimethylformamide)(100 ml) and N-3-bromopropyl succinimide (11.7 g) was added dissolved inabs. DMF (50 ml). NaI (6.5 g) and K₂CO₃ (13.7 g) was added to thereaction mixture and stirred at 110° C. under argon gas for 21 h.Evaporation of DMF and extraction with ethylacetate and citric acid, andbrine, then drying of the organic phase with Na₂SO₄ resulted an oilafter filtration and evaporation. This oil was dissolved in chloroformand poured on a silica gel pad and washed with 50% ethylacetate/hexanes.The pure product solution was evaporated to yield 12.4 g (100%)2-[3-(4-Hydroxy-phenylsulfanyl)-propyl]-isoindole-1,3-dione.

2-[3-(4-Hydroxy-phenylsulfanyl)-propyl]-isoindole-1,3-dione (10.5 g) wasdissolved in ethanol (450 ml) and treated with hydrazine monohydrate (17ml) at 100° C. for 2 h in argon gas. Filtration and evaporation thencrystallization from ethanol gave 4-(3-amino-propylsulfanyl)-phenol (4.2g).

4-(3-Amino-propylsulfanyl)-phenol (4.1 g) was dissolved in abs. CH₂Cl₂and dinitrophenyl-carbonate was added. The reaction was complete afteran overnight stirring at room temperature under argon gas. The solventwas then removed and the crude product was purified by chromatographyresulting [3-(4-hydroxy-phenylsulfanyl)-propyl]-carbamic acid4-nitro-phenyl ester (2 g).

[3-(4-Hydroxy-phenylsulfanyl)-propyl]-carbamic acid 4-nitro-phenyl ester(50 mg) was dissolved in abs. dioxane (3 ml) and added to dry lcaaCPG(long-chain alkylamino controlled pore glass beads available fromAldrich, 450 mg) and triethylamine (20 μl) and was shaken slowly for 1day. The functionalized CPG was washed several times with dioxane, DMF,water, dioxane, to give the product:

This product, hydroxyphenyl-4-sulfanylpropyl-urea-CPG (450 mg), wasreacted with phosgene (5 ml 2M solution in toluene) at room temperaturefor 2 hours in the presence of triethylamine (40 μl) in a Merrifieldflask under argon gas. The solvent and the excess of phosgene wasremoved by suction. The CPG chloroformate was dried under vacuo for 20min, when a solution of 5′-DMT-thymidine (100 mg) in abs. pyridine(freshly distilled from molecular sieves, 5 ml) was added under argon.Shaking overnight, then filtration and washing with methanol,ethylacetate, methanol, acetone and drying resulted in the DMT-T S urealinker functionalized CPG, represented below:

Synthesis of Oligodeoxyribonucleotides and Oligoribonucleotides.

The solid phase synthesis of oligodeoxyribonucleotidesoligoribonucleotides was accomplished using an ABI model 394 automatedDNA synthesizer from Applied Biosystems (Foster City, Calif.). Thesynthesis cycle was adapted from a standard one-micromolar2-cyanoethyl-phosphoramidite RNA or DNA synthesis cycle. For the ACEchemistry a separate synthesizer was specially adapted with Teflontubing and fittings to handle the fluoride ion deblock conditions. TheACE chemistry was performed as described by Scaringe et. al. J. Am.Chem. Soc., 1998, 120(45) 11820-11821. The TOM chemistry was performedas described by Pitsch, et. al. in U.S. Pat. No. 5,986,084. RNA wassynthesized using the 2′-TBDMS method as described by Wincott et. al.,Nucleic Acids Research, 1995, 23, 2677-2684.

Deprotection with Hydrogen Peroxide Solution of Chemically SynthesizedRNA on Peroxyanion Cleavable Linker Containing Commercially AvailableExocyclic Amino Protecting Groups and Commercially Available 2′ HydroxylProtecting Groups Example 1

RNA synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith t-butylphenoxyacetyl (Sinha, et. al., Biochimie, 1993, 75, 13-23).The solid support was polystyrene containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters was cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups were then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 2

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith t-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclicamines. The RNA containing the 2′-TOM protecting groups was thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture was diluted with water and purified by ion-exchangechromatography.

Example 3

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith t-butylphenoxyacetyl. The solid support was the polystyrene basedRapp Polymere containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiesterswere cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This released the RNA oligonucleotides intosolution, deprotected the exocyclic amines. The RNA was then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolated by centrifugation.

Example 4

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith t-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasedthe RNA oligonucleotides into solution, and deprotected the exocyclicamines. The RNA containing the 2′-TBDMS protecting groups was thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture was diluted with water and purified by ion-exchangechromatography.

Example 5

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith t-butylphenoxyacetyl. The solid support was the polystyrene-basedRapp Polymere containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiesterswere cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This released the RNA oligonucleotides intosolution, and deprotected the exocyclic amines. The RNA was thendirectly precipitated by adding 5 volumes of anhydrous ethanol, coolingon dry ice then isolated by centrifugation.

Deprotection of New Amino Protecting Groups (I-X) on ChemicallySynthesized RNA with hydrogen Peroxide Solution

Example 6

RNA synthesized with 2′-ACE monomers. Cytidine is protected with acetyl,Adenosine is protected with isobutyryl, Guanosine is protected withN-(methylthiomethyloxy-carbonyl). The solid support is the polystyrenebased Rapp Polymer containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiestersis cleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclic aminesand modified the 2′-ACE groups. The 2′-ACE groups are then cleaved usinga buffered aqueous formic acid solution at pH 3.8 overnight.

Example 7

RNA synthesized with 2′-ACE monomers. Cytidine is protected with acetyl,Adenosine is protected with isobutyryl, Guanosine is protected withN-(methylthiocarbamate). The solid support is the polystyrene based RappPolymer containing a peroxide oxidizable safety catch linker. Thecapping step using acetic anhydride was removed from the synthesiscycle. Following synthesis, the methyl protecting groups on thephosphodiesters is cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups are then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 8

RNA synthesized with 2′-ACE monomers. Cytidine is protected with acetyl,Adenosine is protected with isobutyryl, Guanosine is protected withN-thiomethylacetyl. The solid support is the polystyrene based RappPolymer containing a peroxide oxidizable safety catch linker. Thecapping step using acetic anhydride was removed from the synthesiscycle. Following synthesis, the methyl protecting groups on thephosphodiesters is cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups are then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 9

RNA synthesized with 2′-ACE monomers. Cytidine is protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), Adenosine isprotected with isobutyryl, Guanosine is protected witht-butylphenoxyacetyl. The solid support is the polystyrene based RappPolymer containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters iscleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclic aminesand modified the 2′-ACE groups. The 2′-ACE groups are then cleaved usinga buffered aqueous formic acid solution at pH 3.8 overnight.

Example 10

RNA synthesized with 2′-ACE monomers. Cytidine is protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), Adenosine is protectedwith isobutyryl, Guanosine is protected with t-butylphenoxyacetyl. Thesolid support is the polystyrene based Rapp Polymer containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters is cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups are then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 11

RNA synthesized with 2′-ACE monomers. Cytidine is protected with acetyl,Adenosine is protected with N-(4-thiomethylbenzoyl), Guanosine isprotected with t-butylphenoxyacetyl. The solid support is thepolystyrene based Rapp Polymer containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters is cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups are then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 12

RNA synthesized with 2′-ACE monomers. Cytidine is protected with acetyl,Adenosine is protected with N-(2-thiomethylbenzoyl), Guanosine isprotected with t-butylphenoxyacetyl. The solid support is thepolystyrene based Rapp Polymer containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters is cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups are then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 13

RNA synthesized with 2′-ACE monomers. Cytidine is protected withN-(2-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support isthe polystyrene based Rapp Polymer containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters is cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups are then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 14

RNA synthesized with 2′-ACE monomers. Cytidine is protected withN-(4-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support isthe polystyrene based Rapp Polymer containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters is cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups are then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 15

RNA synthesized with 2′-ACE monomers. Cytidine is protected with acetyl,Adenosine is protected with isobutyryl, Guanosine is protected withN-(t-butylthiocarbamate). The solid support is the polystyrene basedRapp Polymer containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiestersis cleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclic aminesand modified the 2′-ACE groups. The 2′-ACE groups are then cleaved usinga buffered aqueous formic acid solution at pH 3.8 overnight.

Example 16

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(methylthiomethyloxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support iscontrolled pore glass containing a peroxide oxidizable safety catchlinker. The Acetic Anhydride capping step was removed from the synthesiscycle. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 17

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(methylthiomethyloxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support isthe polystyrene based Rapp Polymere containing a peroxide oxidizablesafety catch linker. The acetic anhydride capping step was removed fromthe synthesis cycle. Following synthesis, the methyl protecting groupson the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 18

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-(methylthiocarbamate). The solid support is controlled pore glasscontaining a peroxide oxidizable safety catch linker. The aceticanhydride capping step was removed from the synthesis cycle. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution, and deprotects the exocyclicamines. The RNA containing the 2′-TOM protecting groups is thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture is diluted with water and purified by ion-exchangechromatography.

Example 19

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-(methylthiocarbamate). The solid support is the polystyrene basedRapp Polymere containing a peroxide oxidizable safety catch linker. Theacetic anhydride capping step was removed from the synthesis cycle.Following synthesis, the methyl protecting groups on the phosphodiestersare cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 20

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-thiomethylacetyl. The solid support is controlled pore glasscontaining a peroxide oxidizable safety catch linker. The aceticanhydride capping step was removed from the synthesis cycle. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution, and deprotects the exocyclicamines. The RNA containing the 2′-TOM protecting groups is thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture is diluted with water and purified by ion-exchangechromatography.

Example 21

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-thiomethylacetyl. The solid support is the polystyrene based RappPolymere containing a peroxide oxidizable safety catch linker. Theacetic anhydride capping step was removed from the synthesis cycle.Following synthesis, the methyl protecting groups on the phosphodiestersare cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 22

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), Adenosine isprotected with isobutyryl, Guanosine is protected witht-butylphenoxyacetyl. The solid support is controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution, and deprotects the exocyclicamines. The RNA containing the 2′-TOM protecting groups is thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture is diluted with water and purified by ion-exchangechromatography.

Example 23

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), Adenosine isprotected with isobutyryl, Guanosine is protected witht-butylphenoxyacetyl. The solid support is the polystyrene based RappPolymere containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a solution of HF/tetraethylene diamine (20% TEMED, 10%HF(aq) in acetonitrile at pH 8.6) for 2 hours at room temperature. Thefluoride ion solution is washed from the support using acetonitrilefollowed by water. The support is then treated with a 6% hydrogenperoxide solution buffered at pH 9.4 using aminomethylpropanol buffer in10/90 ethanol/water for 4 hours. This releases the RNA oligonucleotidesinto solution, and deprotects the exocyclic amines. The RNA is thendirectly precipitated by adding 5 volumes of anhydrous ethanol, coolingon dry ice then isolating by centrifugation.

Example 24

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), Adenosine is protectedwith isobutyryl, Guanosine is protected with t-butylphenoxyacetyl. Thesolid support is controlled pore glass containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 25

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), Adenosine is protectedwith isobutyryl, Guanosine is protected with t-butylphenoxyacetyl. Thesolid support is the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 26

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with N-(4-thiomethylbenzoyl), Guanosineis protected with t-butylphenoxyacetyl. The solid support is controlledpore glass containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiestersare cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 27

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with N-(4-thiomethylbenzoyl), Guanosineis protected with t-butylphenoxyacetyl. The solid support is thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 28

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with N-(2-thiomethylbenzoyl), Guanosineis protected with t-butylphenoxyacetyl. The solid support is controlledpore glass containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiestersare cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 29

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with N-(2-thiomethylbenzoyl), Guanosineis protected with t-butylphenoxyacetyl. The solid support is thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 30

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(2-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support iscontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 31

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(2-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support isthe polystyrene based Rapp Polymere containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 32

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(4-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support iscontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TOM protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 33

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withN-(4-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support isthe polystyrene based Rapp Polymere containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 34

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-(t-butylthiocarbamate). The solid support is controlled poreglass containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution, and deprotects the exocyclicamines. The RNA containing the 2′-TOM protecting groups is thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture is diluted with water and purified by ion-exchangechromatography.

Example 35

RNA is synthesized using 2′-TOM monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-(t-butylthiocarbamate). The solid support is the polystyrenebased Rapp Polymere containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 36

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(methylthiomethyloxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support iscontrolled pore glass containing a peroxide oxidizable safety catchlinker. The Acetic Anhydride capping step was removed from the synthesiscycle. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 37

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(methylthiomethyloxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support isthe polystyrene based Rapp Polymere containing a peroxide oxidizablesafety catch linker. The acetic anhydride capping step was removed fromthe synthesis cycle. Following synthesis, the methyl protecting groupson the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 38

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-(methylthiocarbamate). The solid support is controlled pore glasscontaining a peroxide oxidizable safety catch linker. The aceticanhydride capping step was removed from the synthesis cycle. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution, and deprotects the exocyclicamines. The RNA containing the 2′-TBDMS protecting groups is thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture is diluted with water and purified by ion-exchangechromatography.

Example 39

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-(methylthiocarbamate). The solid support is the polystyrene basedRapp Polymere containing a peroxide oxidizable safety catch linker. Theacetic anhydride capping step was removed from the synthesis cycle.Following synthesis, the methyl protecting groups on the phosphodiestersare cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 40

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-thiomethylacetyl. The solid support is controlled pore glasscontaining a peroxide oxidizable safety catch linker. The aceticanhydride capping step was removed from the synthesis cycle. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution, and deprotects the exocyclicamines. The RNA containing the 2′-TBDMS protecting groups is thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture is diluted with water and purified by ion-exchangechromatography.

Example 41

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-thiomethylacetyl. The solid support is the polystyrene based RappPolymere containing a peroxide oxidizable safety catch linker. Theacetic anhydride capping step was removed from the synthesis cycle.Following synthesis, the methyl protecting groups on the phosphodiestersare cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 42

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), Adenosine isprotected with isobutyryl, Guanosine is protected witht-butylphenoxyacetyl. The solid support is controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution, and deprotects the exocyclicamines. The RNA containing the 2′-TBDMS protecting groups is thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture is diluted with water and purified by ion-exchangechromatography.

Example 43

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane), Adenosine isprotected with isobutyryl, Guanosine is protected witht-butylphenoxyacetyl. The solid support is the polystyrene based RappPolymere containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a solution of HF/tetraethylene diamine (20% TEMED, 10%HF(aq) in acetonitrile at pH 8.6) for 2 hours at room temperature. Thefluoride ion solution is washed from the support using acetonitrilefollowed by water. The support is then treated with a 6% hydrogenperoxide solution buffered at pH 9.4 using aminomethylpropanol buffer in10/90 ethanol/water for 4 hours. This releases the RNA oligonucleotidesinto solution, and deprotects the exocyclic amines. The RNA is thendirectly precipitated by adding 5 volumes of anhydrous ethanol, coolingon dry ice then isolating by centrifugation.

Example 44

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), Adenosine is protectedwith isobutyryl, Guanosine is protected with t-butylphenoxyacetyl. Thesolid support is controlled pore glass containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 45

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(carbonyloxy-1-methylthiomethylcyclohexane), Adenosine is protectedwith isobutyryl, Guanosine is protected with t-butylphenoxyacetyl. Thesolid support is the polystyrene based Rapp Polymere containing aperoxide oxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 46

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with N-(4-thiomethylbenzoyl), Guanosineis protected with t-butylphenoxyacetyl. The solid support is controlledpore glass containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiestersare cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 47

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with N-(4-thiomethylbenzoyl), Guanosineis protected with t-butylphenoxyacetyl. The solid support is thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 48

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with N-(2-thiomethylbenzoyl), Guanosineis protected with t-butylphenoxyacetyl. The solid support is controlledpore glass containing a peroxide oxidizable safety catch linker.Following synthesis, the methyl protecting groups on the phosphodiestersare cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 49

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with N-(2-thiomethylbenzoyl), Guanosineis protected with t-butylphenoxyacetyl. The solid support is thepolystyrene based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 50

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(2-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support iscontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 51

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(2-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support isthe polystyrene based Rapp Polymere containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,l-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 52

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(4-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support iscontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This releases theRNA oligonucleotides into solution, and deprotects the exocyclic amines.The RNA containing the 2′-TBDMS protecting groups is then precipitatedfrom the hydrogen peroxide solution and exposed to a solution ofHF/tetraethylene diamine (20% TEMED, 10% HF(aq) in acetonitrile at pH8.6) for 6 hours at room temperature. The reaction mixture is dilutedwith water and purified by ion-exchange chromatography.

Example 53

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withN-(4-thiomethylphenoxycarbonyl), Adenosine is protected with isobutyryl,Guanosine is protected with t-butylphenoxyacetyl. The solid support isthe polystyrene based Rapp Polymere containing a peroxide oxidizablesafety catch linker. Following synthesis, the methyl protecting groupson the phosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Example 54

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-(t-butylthiocarbamate). The solid support is controlled poreglass containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters arecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution is washedfrom the solid support bound oligonucleotide using water. The support isthen treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasesthe RNA oligonucleotides into solution, and deprotects the exocyclicamines. The RNA containing the 2′-TBDMS protecting groups is thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture is diluted with water and purified by ion-exchangechromatography.

Example 55

RNA is synthesized using 2′-TBDMS monomers. Cytidine is protected withacetyl, Adenosine is protected with isobutyryl, Guanosine is protectedwith N-(t-butylthiocarbamate). The solid support is the polystyrenebased Rapp Polymere containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support is then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This releases the RNA oligonucleotides intosolution, and deprotects the exocyclic amines. The RNA is then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolating by centrifugation.

Deprotection with Hydrogen Peroxide Solution of Chemically SynthesizedRNA on Peroxyanion Cleavable Linker Containing Commercially AvailableExocyclic Amino Protecting Groups and Novel 2′ Hydroxyl ProtectingGroups Example 1

RNA synthesized with 2′-ACE monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith tert-butylphenoxyacetyl (Sinha, et. al., Biochimie, 1993, 75,13-23). The solid support was polystyrene containing a peroxideoxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters was cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 50/50 methanol/water. This released theRNA oligonucleotides into solution, deprotected the exocyclic amines andmodified the 2′-ACE groups. The 2′-ACE groups were then cleaved using abuffered aqueous formic acid solution at pH 3.8 overnight.

Example 2

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith tert-butylphenoxyacetyl. The solid support was controlled poreglass containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclicamines. The RNA containing the 2′-TOM protecting groups was thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture was diluted with water and purified by ion-exchangechromatography.

Example 3

RNA was synthesized using 2′-TOM monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith tert-butylphenoxyacetyl. The solid support was the polystyrenebased Rapp Polymere containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution was washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This released the RNA oligonucleotides intosolution, deprotected the exocyclic amines. The RNA was then directlyprecipitated by adding 5 volumes of anhydrous ethanol, cooling on dryice then isolated by centrifugation.

Example 4

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith tert-butylphenoxyacetyl. The solid support was controlled poreglass containing a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 50/50 methanol/water. This releasedthe RNA oligonucleotides into solution, and deprotected the exocyclicamines. The RNA containing the 2′-TBDMS protecting groups was thenprecipitated from the hydrogen peroxide solution and exposed to asolution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 6 hours at room temperature. The reactionmixture was diluted with water and purified by ion-exchangechromatography.

Example 5

RNA was synthesized using 2′-TBDMS monomers. Cytidine was protected withacetyl, Adenosine was protected with isobutyryl, Guanosine was protectedwith tert-butylphenoxyacetyl. The solid support was thepolystyrene-based Rapp Polymere containing a peroxide oxidizable safetycatch linker. Following synthesis, the methyl protecting groups on thephosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a solution of HF/tetraethylene diamine (20% TEMED, 10% HF(aq) inacetonitrile at pH 8.6) for 2 hours at room temperature. The fluorideion solution is washed from the support using acetonitrile followed bywater. The support was then treated with a 6% hydrogen peroxide solutionbuffered at pH 9.4 using aminomethylpropanol buffer in 10/90ethanol/water for 4 hours. This released the RNA oligonucleotides intosolution, and deprotected the exocyclic amines. The RNA was thendirectly precipitated by adding 5 volumes of anhydrous ethanol, coolingon dry ice then isolated by centrifugation.

Example 6

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with acetyl, Adenosine was protectedwith isobutyryl, Guanosine was protected with tert-butylphenoxyacetyl.The solid support was controlled pore glass containing a peroxideoxidizable safety catch linker. Following synthesis, the methylprotecting groups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This released the RNAoligonucleotides into solution, deprotected the exocyclic amines, andthe 2′-BSC groups. The RNA was then directly precipitated by adding 5volumes of anhydrous ethanol, cooling on dry ice then isolated bycentrifugation.

Example 7

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with acetyl, Adenosine was protectedwith isobutyryl, Guanosine was protected withN-(methylthiomethyloxycarbonyl). The solid support is controlled poreglass containing a peroxide oxidizable safety catch linker. Aceticanhydride capping was removed from the synthesis cycle. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclicamines, and the 2′-BSC groups. The RNA was then directly precipitated byadding 5 volumes of anhydrous ethanol, cooling on dry ice then isolatedby centrifugation.

Example 8

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with acetyl, Adenosine was protectedwith isobutyryl, Guanosine was protected with N-(methylthiocarbamate).The solid support was controlled pore glass containing a peroxideoxidizable safety catch linker. Acetic anhydride capping was removedfrom the synthesis cycle. Following synthesis, the methyl protectinggroups on the phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This released the RNAoligonucleotides into solution, deprotected the exocyclic amines and the2′-BSC groups. The RNA was then directly precipitated by adding 5volumes of anhydrous ethanol, cooling on dry ice then isolated bycentrifugation.

Example 9

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with acetyl, Adenosine was protectedwith isobutyryl, Guanosine was protected with N-thiomethylacetyl. Thesolid support was controlled pore glass containing a peroxide oxidizablesafety catch linker. Acetic anhydride capping was removed from thesynthesis cycle. Following synthesis, the methyl protecting groups onthe phosphodiesters were cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution was washed from the solidsupport bound oligonucleotide using water. The support was then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This released the RNAoligonucleotides into solution, deprotected the exocyclic amines and the2′-BSC groups. The RNA was then directly precipitated by adding 5volumes of anhydrous ethanol, cooling on dry ice then isolated bycentrifugation.

Example 10

RNA is synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine is protected with acetyl, Adenosine is protected withisobutyryl, Guanosine is protected withN-(carbonyloxy-1-phethylthiomethyl-1-H-isobutane). The solid support iscontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines and the2′-BSC groups. The RNA is then directly precipitated by adding 5 volumesof anhydrous ethanol, cooling on dry ice then isolating bycentrifugation.

Example 11

RNA is synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with acetyl, Adenosine is protectedwith isobutyryl, Guanosine is protected withN-(carbonyloxy-1-methylthiomethylcyclohexane). The solid support iscontrolled pore glass containing a peroxide oxidizable safety catchlinker. Following synthesis, the methyl protecting groups on thephosphodiesters are cleaved using 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMFfor 30 minutes. The deprotection solution is washed from the solidsupport bound oligonucleotide using water. The support is then treatedwith a 6% hydrogen peroxide solution buffered at pH 9.4 usingaminomethylpropanol buffer in 10/90 ethanol/water. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines and the2′-BSC groups. The RNA is then directly precipitated by adding 5 volumesof anhydrous ethanol, cooling on dry ice then isolating bycentrifugation.

Example 12

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with acetyl, Adenosine was protectedwith N-(4-thiomethyl-benzoyl), Guanosine was protected withtert-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclic aminesand the 2′-BSC groups. The RNA was then directly precipitated by adding5 volumes of anhydrous ethanol, cooling on dry ice then isolated bycentrifugation.

Example 13

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with acetyl, Adenosine was protectedwith N-(2-thiomethyl-benzoyl), Guanosine was protected withtert-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclic aminesand the 2′-BSC groups. The RNA was then directly precipitated by adding5 volumes of anhydrous ethanol, cooling on dry ice then isolated bycentrifugation.

Example 14

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with N-(2-thiomethylphenoxycarbonyl),Adenosine was protected with isobutyryl, Guanosine was protected withtert-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclic aminesand the 2′-BSC groups. The RNA was then directly precipitated by adding5 volumes of anhydrous ethanol, cooling on dry ice then isolated bycentrifugation.

Example 15

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with N-(4-thiomethylphenoxycarbonyl),Adenosine was protected with isobutyryl, Guanosine was protected withtert-butylphenoxyacetyl. The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclic aminesand the 2′-BSC groups. The RNA was then directly precipitated by adding5 volumes of anhydrous ethanol, cooling on dry ice then isolated bycentrifugation.

Example 16

RNA was synthesized using 2′-tert-butylthiocarbonate (BSC) protectedmonomers. Cytidine was protected with acetyl, Adenosine was protectedwith isobutyryl, Guanosine was protected withN-(tert-butylthiocarbamate). The solid support was controlled pore glasscontaining a peroxide oxidizable safety catch linker. Followingsynthesis, the methyl protecting groups on the phosphodiesters werecleaved using 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolatetrihydrate in DMF for 30 minutes. The deprotection solution was washedfrom the solid support bound oligonucleotide using water. The supportwas then treated with a 6% hydrogen peroxide solution buffered at pH 9.4using aminomethylpropanol buffer in 10/90 ethanol/water. This releasedthe RNA oligonucleotides into solution, deprotected the exocyclic aminesand the 2′-BSC groups. The RNA was then directly precipitated by adding5 volumes of anhydrous ethanol, cooling on dry ice then isolated bycentrifugation.

While the foregoing embodiments of the invention have been set forth inconsiderable detail for the purpose of making a complete disclosure ofthe invention, it will be apparent to those of skill in the art thatnumerous changes may be made in such details without departing from thespirit and the principles of the invention. Accordingly, the inventionshould be limited only by the following claims.

All patents, patent applications, and publications mentioned herein arehereby incorporated by reference in their entireties.

1. A method comprising: contacting a polynucleotide bound to a substratevia a cleavable linker with a solution comprising an α-effectnucleophile to result in cleavage of the polynucleotide from thesubstrate; wherein the cleavable linker has a structure selected fromstructures (I), (II), (III), (IV), or (V):

wherein: R3, R4, R5, R6, R7, R10, and R11 are each independentlyselected from H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl,thio, mercapto, amino, amido, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, or -Lnk-Sub, wherein Lnk is a linking group and Sub denotes thesite at which the substrate is attached to the cleavable linker,provided that one and only one of R3, R4, R5, R6, R7, R10, or R11 is-Lnk-Sub; and RPN denotes the site at which the polynucleotide isattached to the cleavable linker;

wherein: R3, R4, R5, R6, R7, R10, and R11 are each independentlyselected from H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl,thio, mercapto, amino, amido, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, or -Lnk-Sub, wherein Lnk is a linking group and Sub denotes thesite at which the substrate is attached to the cleavable linker,provided that one and only one of R3, R4, R5, R6, R7, R10, or R11 is-Lnk-Sub; and RPN denotes the site at which the polynucleotide isattached to the cleavable linker;

wherein: R4, R5, R6, and R7 are each independently selected from H,lower alkyl, modified lower alkyl, thioalkyl, hydroxyl, thio, mercapto,amino, amido, imino, halo, cyano, nitro, nitroso, azido, carboxy,sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, boronyl, or-Lnk-Sub, wherein Lnk is a linking group and Sub denotes the site atwhich the substrate is attached to the cleavable linker, provided thatone and only one of R4, R5, R6, or R7 is -Lnk-Sub; R13 is selected fromlower alkyl, modified lower alkyl, alkyl, or modified alkyl, or aryl;and RPN denotes the site at which the polynucleotide is attached to thecleavable linker;

wherein: R4, R5, R6, R7, R10, and R11 are each independently selectedfrom H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl, thio,mercapto, amino, amido, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, or -Lnk-Sub, wherein Lnk is a linking group and Sub denotes thesite at which the substrate is attached to the cleavable linker,provided that one and only one of R4, R5, R6, R7, R10, or R11 is-Lnk-Sub; R13 is selected from lower alkyl, modified lower alkyl, alkyl,or modified alkyl, or aryl; and RPN denotes the site at which thepolynucleotide is attached to the cleavable linker;

wherein: R4, R5, R6, R7, R10, and R11 are each independently selectedfrom H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl, thio,mercapto, amino, amido, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, or -Lnk-Sub, wherein Lnk is a linking group and Sub denotes thesite at which the substrate is attached to the cleavable linker,provided that one and only one of R4, R5, R6, R7, R10, or R11 is-Lnk-Sub; R13 is selected from lower alkyl, modified lower alkyl, alkyl,or modified alkyl, or aryl; and RPN denotes the site at which thepolynucleotide is attached to the cleavable linker.
 2. The method ofclaim 1 wherein the solution is at a pH of about 6 to about
 12. 3. Themethod of claim 1 wherein the α-effect nucleophile is characterized ashaving a pKa in the range of about 4 to
 13. 4. The method of claim 1wherein the solution comprising the α-effect nucleophile is a solutioncomprising one or more species selected from hydrogen peroxide, aperacid, a perboric acid, an alkylperoxide, a hydroperoxide, abutylhydroperoxide, a benzylhydroperoxide, a phenylhydroperoxide, acumene hydroperoxide, performic acid, a peracetic acid, perbenzoic acid,a substituted perbenzoic acid, chloroperbenzoic acid, perbutyric acid,tertiary-butylperoxybenzoic acid, decanediperoxoic acid, correspondingsalts of said species, and combinations thereof.
 5. The method of claim1 wherein the solution comprising the α-effect nucleophile is a solutioncomprising one or more species selected from hydrogen peroxide, salts ofhydrogen peroxide, and mixtures of hydrogen peroxide and performic acid.6. The method of claim 1 wherein the α-effect nucleophile is formed insitu by a reaction of hydrogen peroxide and a carboxylic acid orcarboxylic acid salt.
 7. The method of claim 1 wherein the cleavablelinker has a structure selected from structures (I) or (II); wherein R3,R4, R6, R7, R10, and R11 are each independently selected from H or loweralkyl; and R5 is -Lnk-Sub.
 8. The method of claim 1 wherein thecleavable linker has a structure selected from structures (III), (IV) or(V); wherein R4, R6, R7, R10, and R11 are each independently selectedfrom H or lower alkyl; R5 is -Lnk-Sub; and R13 is lower alkyl.
 9. Themethod of claim 1, wherein the linking group Lnk is selected from: (1) alower alkyl group; (2) a modified lower alkyl group in which one or morelinkages selected from ether-, thio-, amino-, oxo-, ester-, and amido-is present; (3) a modified lower alkyl substituted with one or moregroups including lower alkyl; aryl, aralkyl, alkoxyl, thioalkyl,hydroxyl, amino, amido, sulfonyl, halo; or (4) a modified lower alkylsubstituted with one or more groups including lower alkyl; alkoxyl,thioalkyl, hydroxyl, amino, amido, sulfonyl, halo, and in which one ormore linkages selected from ether-, thio-, amino-, oxo-, ester-, andamido- is present.
 10. The method of claim 1, wherein the linking groupLnk has the structure—(CH2)m-Lkg-(CH2)n- wherein: m and n are integers independently selectedfrom the range of 1 to about 12, and Lkg is a linkage selected fromether-, thio-, amino-, oxo-, ester-, or amido-.
 11. The method of claim1, further comprising recovering the polynucleotide after cleavage ofthe polynucleotide from the substrate.
 12. The method of claim 1,wherein the polynucleotide comprises at least one protecting groupselected from a nucleobase protecting group, a 2′-hydroxyl protectinggroup, and a phosphate protecting group, wherein said at least oneprotecting group is labile under conditions which include an α-effectnucleophile; and wherein said contacting results in concurrent cleavageof the polynucleotide from the substrate and deprotection of thepolynucleotide.
 13. The method of claim 1: wherein the polynucleotidebound to the substrate via the cleavable linker is an RNA, said RNA hasa 2′-hydroxyl protecting group, said 2′hydroxyl protecting group ischaracterized as being peroxyanion-labile; wherein the α-effectnucleophile is a peroxyanion; and wherein said contacting results inconcurrent cleavage of the RNA from the substrate and cleavage of the2′-hydroxyl protecting group.
 14. The method of claim 1: wherein thepolynucleotide comprises a 2′-hydroxyl protecting group and at least oneadditional protecting group selected from a nucleobase protecting groupand a phosphorus protecting group; wherein said 2′-hydroxyl protectinggroup is stable under conditions which include an α-effect nucleophile;wherein said at least one additional protecting group is labile underconditions which include an α-effect nucleophile; and wherein saidcontacting results in concurrent cleavage of the polynucleotide from thesubstrate and cleavage of said at least one additional protecting group.15. The method of claim 14 further comprising cleaving the 2′hydroxylprotecting group under conditions sufficient to result in cleavage ofthe 2′hydroxyl protecting group, wherein said conditions do not includeα-effect nucleophile.
 16. The method of claim 1: wherein thepolynucleotide comprises a 2′-hydroxyl protecting group, a phosphorusprotecting group, and a nucleobase protecting group; wherein said2′-hydroxyl protecting group and said phosphorus protecting group arestable under conditions which include an α-effect nucleophile; whereinsaid nucleobase protecting group is labile under conditions whichinclude an α-effect nucleophile; and wherein said contacting results inconcurrent cleavage of the polynucleotide from the substrate andcleavage of said nucleobase protecting group.
 17. The method of claim 16further comprising cleaving at least one protecting group selected fromthe 2′-hydroxyl protecting group and the phosphorus protecting groupunder conditions sufficient to result in cleavage of the at least oneprotecting group, wherein said conditions do not include α-effectnucleophile.
 18. A composition comprising a polynucleotide bound to asubstrate via a cleavable linker, wherein the cleavable linker has astructure selected from structures (I), (II), (III), (IV), or (V):

wherein: R3, R4, R5, R6, R7, R10, and R11 are each independentlyselected from H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl,thio, mercapto, amino, amido, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, or -Lnk-Sub, wherein Lnk is a linking group and Sub denotes thesite at which the substrate is attached to the cleavable linker,provided that one and only one of R3, R4, R5, R6, R7, R10, or R11 is-Lnk-Sub; and RPN denotes the site at which the polynucleotide isattached to the cleavable linker;

wherein: R3, R4, R5, R6, R7, R10, and R11 are each independentlyselected from H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl,thio, mercapto, amino, amido, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, or -Lnk-Sub, wherein Lnk is a linking group and Sub denotes thesite at which the substrate is attached to the cleavable linker,provided that one and only one of R3, R4, R5, R6, R7, R10, or R11 is-Lnk-Sub; and RPN denotes the site at which the polynucleotide isattached to the cleavable linker;

wherein: R4, R5, R6, and R7 are each independently selected from H,lower alkyl, modified lower alkyl, thioalkyl, hydroxyl, thio, mercapto,amino, amido, imino, halo, cyano, nitro, nitroso, azido, carboxy,sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, boronyl, or-Lnk-Sub, wherein Lnk is a linking group and Sub denotes the site atwhich the substrate is attached to the cleavable linker, provided thatone and only one of R4, R5, R6, or R7 is -Lnk-Sub; R13 is selected fromlower alkyl, modified lower alkyl, alkyl, or modified alkyl, or aryl;and RPN denotes the site at which the polynucleotide is attached to thecleavable linker;

wherein: R4, R5, R6, R7, R10, and R11 are each independently selectedfrom H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl, thio,mercapto, amino, amido, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, or -Lnk-Sub, wherein Lnk is a linking group and Sub denotes thesite at which the substrate is attached to the cleavable linker,provided that one and only one of R4, R5, R6, R7, R10, or R11 is-Lnk-Sub; R13 is selected from lower alkyl, modified lower alkyl, alkyl,or modified alkyl, or aryl; and RPN denotes the site at which thepolynucleotide is attached to the cleavable linker;

wherein: R4, R5, R6, R7, R10, and R11 are each independently selectedfrom H, lower alkyl, modified lower alkyl, thioalkyl, hydroxyl, thio,mercapto, amino, amido, imino, halo, cyano, nitro, nitroso, azido,carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy,boronyl, or -Lnk-Sub, wherein Lnk is a linking group and Sub denotes thesite at which the substrate is attached to the cleavable linker,provided that one and only one of R4, R5, R6, R7, R10, or R11 is-Lnk-Sub; R13 is selected from lower alkyl, modified lower alkyl, alkyl,or modified alkyl, or aryl; and RPN denotes the site at which thepolynucleotide is attached to the cleavable linker.
 19. The compositionof claim 18 wherein the cleavable linker is characterized as being aperoxyanion-labile linker.
 20. The composition of claim 18 wherein thecleavable linker has a structure selected from structures (I) or (II);wherein R3, R4, R6, R7, R10, and R11 are each independently selectedfrom H or lower alkyl; and R5 is -Lnk-Sub.
 21. The composition of claim18 wherein the cleavable linker has a structure selected from structures(III), (IV) or (V); wherein R4, R6, R7, R10, and R11 are eachindependently selected from H or lower alkyl; R5 is -Lnk-Sub; and R13 islower alkyl.
 22. The composition of claim 18, wherein the linking groupLnk is selected from: (1) a lower alkyl group; (2) a modified loweralkyl group in which one or more linkages selected from ether-, thio-,amino-, oxo-, ester-, and amido- is present; (3) a modified lower alkylsubstituted with one or more groups including lower alkyl; aryl,aralkyl, alkoxyl, thioalkyl, hydroxyl, amino, amido, sulfonyl, halo; or(4) a modified lower alkyl substituted with one or more groups includinglower alkyl; alkoxyl, thioalkyl, hydroxyl, amino, amido, sulfonyl, halo,and in which one or more linkages selected from ether-, thio-, amino-,oxo-, ester-, and amido- is present.
 23. The composition of claim 18,wherein the linking group Lnk has the structure—(CH2)m-Lkg-(CH2)n- wherein: m and n are integers independently selectedfrom the range of 1 to about 12, and Lkg is a linkage selected fromether-, thio-, amino-, oxo-, ester-, or amido-.
 24. The composition ofclaim 18 wherein the polynucleotide comprises at least one protectinggroup selected from a nucleobase protecting group, a 2′-hydroxylprotecting group, and a phosphorus protecting group, wherein said atleast one protecting group and the cleavable linker are bothcharacterized as labile under conditions which include an α-effectnucleophile.
 25. The composition of claim 18: wherein the polynucleotidebound to the substrate via the cleavable linker is an RNA, said RNA hasa 2′-hydroxyl protecting group; and wherein said 2′hydroxyl protectinggroup and the cleavable linker are characterized as being labile underconditions which include an α-effect nucleophile.
 26. The composition ofclaim 18: wherein the polynucleotide comprises a 2′-hydroxyl protectinggroup and at least one additional protecting group selected from anucleobase protecting group and a phosphorus protecting group, whereinsaid 2′-hydroxyl protecting group is characterized as stable underconditions which include an α-effect nucleophile; wherein said at leastone additional protecting group is characterized as labile underconditions which include an α-effect nucleophile.
 27. The composition ofclaim 18: wherein the polynucleotide comprises a 2′-hydroxyl protectinggroup, a phosphorus protecting group, and a nucleobase protecting group,wherein said 2′-hydroxyl protecting group is characterized as stableunder conditions which include an α-effect nucleophile; wherein saidphosphate protecting group is characterized as stable under conditionswhich include an α-effect nucleophile; and wherein said nucleobaseprotecting group is characterized as labile under conditions whichinclude an α-effect nucleophile.